Method of making printed reagent test devices

ABSTRACT

A test device for determining the presence of a constituent in a sample, and a method for making it are disclosed. The test device comprises reactants (e.g. reagents, enzymes, etc.) incorporated with a carrier matrix such that when the device is wetted with a test sample, the reactants and the constituent react to produce a detectable response. The reactants are positioned separately from each other on the matrix in substantially, discrete, non-contacting areas. Hence, reactants are maintained substantially separate from each other until the test device is wetted with the sample.

RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.779,824, filed Mar. 21, 1977, now abandoned which is, in turn, adivisional application of U.S. application Ser. No. 701,403, filed June20, 1976 now U.S. Pat. No. 4,046,513.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a test device for determining thepresence of a constituent in a sample. The invention as defined by theclaims comprises a test device, and a method for preparing it, in whichpotentially incompatible reactants are kept separate from each otheruntil the actual testing of a sample, such as a bodily fluid, takesplace. Hence, the device comprises a carrier matrix incorporated with atleast two reactants capable of interacting with a sample constituentbeing analyzed to produce a detectable response. The reactants arepositioned separately from each other in substantially discrete,noncontacting areas on the carrier matrix.

2. Description of the Prior Art

The burgeoning field of test devices in the form of test strips hasproviding convenient and rapid analysis of various types of samples,including samples of biological, industrial, and automotive fluids, andthe like. Diagnostic devices designed for detecting various clinicallysignificant substances or constituents in biological fluids, such asurine and blood, including lysed or unlysed blood, blood plasma, andblood serum, have in many cases supplanted prior wet chemistrytechniques which were both cumbersome and time-consuming. Thesediagnostic devices have thus assisted in the fast and accurate diagnosisand treatment of disease.

Conventional test strips generally comprise an absorbent or porousmatrix incorporated with indicator reactants, usually of a colorimetrictype. The sample to be tested is contacted with the matrix, such as bymomentary immersion where the sample is liquid, and the indicatorresponse is observed after a set period of time. For example, in areagent strip for the detection of occult blood in urine a diagnosticstrip can be employed which comprises an absorbent paper impregnatedwith o-tolidine and a peroxide. When this strip is wetted with urinecontaining occult blood, decomposition of the peroxide occurs with theaccompanying oxidation of the o-tolidine to produce a color response.This test is sensitive and extremely useful in diagnosing urinary tractdisorders. However, because of the relative incompatibility of employedreactants, shelf life has often been found to be relatively short andthe strips can lose their sensitivity after long periods of storage.

Similar problems of reactant incompatibility occur in many other typesof strips where more than one chemical reaction is involved. Forexample, reactants for testing ketone, blood urea nitrogen (BUN), andgalactose levels have been known to have limited shelf lives. In orderto explore ways of extending the shelf like of reagent test strips, i.e.methods of reducing the relative incompatibility of reactants,experiments were conducted to determine whether it would be possible tophysically separate incompatible reagents on the strip itself. Prior tothis work the successful separation of incompatible reagents had notbeen reported. The experiments were successful and it was found thatreagent strips could indeed be prepared in which imcompatible reactantswere physically separated until becoming contacted with the sample to beanalyzed. Strips prepared in accordance with the present invention haveexcellent shelf life and are vastly superior in this respect to presentcommercial strips containing the same reactants.

SUMMARY OF THE INVENTION

The present invention relates to a test device for detecting aconstituent in a sample, particularly a bodily fluid. The devicecomprises a carrier matrix incorporated with at least two differentreactants capable of mutually interacting in the presence of theconstituent to produce a detectable response, the different reactantsbeing physically separated from each other on the matrix. The differentreactants are printed separately on the matrix as a plurality ofsubstantially discrete areas, including dots, microdots, lines or thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

Other and further advantages and features of the invention will beapparent to those skilled in the art from the following detaileddescription thereof, taken in conjunction with the accompanyingdrawings, in which:

FIGS. 1 through 5 are diagrammatic illustrations of different patternsfor applying reactants-containing inks in accordance with the presentinvention. All of the FIGS. depict cases where at least two inks areemployed, FIG. 4 showing the use of three reactant inks. Thus, FIGS. 1,2 and 3 depict first and second reactant inks printed on a carriermatrix in first and second arrays of impressions, respectively. Theimpressions, or dots, of each array are shown interspersed, one with theother, in a substantially non-contacting spatial arrangement. FIG. 4shows a 3-ink system printed in the same interspersed, substantiallynoncontacting arrangement. FIG. 5 portrays a case where the impressionswhich form first and second arrays of reactant inks are parallel lines.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention at least two reactant materialsare arranged in separate arrays of impressions on a matrix. Circulardots represent an optimum shape for the impressions from the standpointof packing density and reactant diffusions during an assay. The dots canbe printed so as not to touch each other and dissimilar dots can bepresent in equal or unequal numbers as desired. Printed lines of varyingwidths and other impressions such as special symbols can be substitutedfor dots if desired. Moreover, an array can comprise impressions whichdiffer in both shape and spatial arrangement. For example, impressionsof one reactant ink can be spatially arranged more densely at oneportion of the array than at another. Moreover the interspersion ofnon-contacting impressions of two inks need not be uniform or regular.

Referring to FIG. 1, one reactant is depicted as white dots, the secondreactant being depicted as interspersed black dots. However, thisarrangement is not the densest dot arrangement. Dots of the samereactant can be placed in a line (as A and B in FIG. 2) or can alternateon a line (as A and B in FIG. 3). FIG. 4 depicts a further pattern whichis suitable where three different reactants (A, B and C) are employed.In FIG. 4 any reactant dot has six nearest neighbors which comprisethree dots of each of the other two reactants.

Another useful technique in separating the printed reactants is theapplication of recurring stripes to a carrier matrix. Hence, for a tworeactant system, alternate stripes of the complimentary reactants can belaid out parallel on a matrix as in FIG. 5, in which solid recurringlines 1 represent a first reactant and dotted recurring lines 2represent a second reactant.

The present invention has also been found to exhibit great utility inthe deposition of enzymes entrapped in a polymer matrix. Hence, enzymessuch as glucose oxidase can be printed in an ink containing acrylamideand a photopolymerization initiator such as potassium persulfate. Suchan ink could be printed as dots, stripes, or other configurations andexposed after printing with a Number 2 photo flood lamp to effectpolymerization.

In one embodiment of this particular entrapping technique, a stocksolution of monomer compound is prepared by dissolving 40 g ofacrylamide in 100 ml of 0.1 M phosphate buffer having a pH of 7.4. Across linking reactant is prepared by dissolving 2.3 g ofN,N-methylenebisacrylamide in 100 ml of 0.1 M phosphate buffer (pH 7.4).Gels can then be prepared by mixing these solutions in desiredproportions together with a solution of an enzyme such as glucoseoxidase. This gel can then be printed onto an inert matrix in accordancewith the present invention and cured in situ.

Other applications of entrapped enzymes will be easily ascertainable byone skilled in the art by reference to U.S. Pat. Nos. 3,788,950,3,793,445, 3,841,971, 3,859,169, and 3,935,071. These and otherreferences in the prior art discuss in detail the physical entrapment ofenzymes as well as enzymes covalently bound to a substrate and suchtechniques are readily applicable to the present invention.

In each of the above embodiments, whether dots or stripes, it can beseen that when the test device is wetted the separated reactants will atthat time combine and interact with the constituent being analyzed.Typical test reactant compositions are set forth in U.S. Pat. Nos.3,438,737; 3,095,277; 3,212,855; 3,164,534; 3,050,373; 2,981,606;3,123,443; 3,252,762; 3,290,117; 3,092,463; 3,012,976; 3,122,420;3,453,180; 3,585,001; 3,585,004; and 3,447,905 which are incorporatedherein by reference.

The test device of the present invention is optimally prepared usingprinting techniques. For example, a polystyrene matrix can be printedwith a first reactant ink and subsequently printed with a secondreactant ink such that the first and second inks are imprinted insubstantially non-contacting, coplanar impressions. Thus, in the case ofan occult blood test device, a first ink containing o-tolidine is silkscreened as a plurality of dots onto a polystyrene matrix. Subsequently,a second plurality of dots, juxtaposed with the first, is silk screenedonto the matrix. The second plurality comprises a reagent ink containinga peroxide such as cumene hydroperoxide.

In another embodiment of the present invention, offset printingtechniques are employed. An example of how this technique is employed inthe present invention is the use of a rubber stamp containing raiseddots. The rubber stamp is inked with a first reactant ink and applied tothe matrix leaving a dot impression of the first reactant. The secondreactant is stamped similarly onto the matrix except that the dots arejuxtaposed with those of the first reactants.

Still another technique of preparing the present test device is that ofapplying ribbons or stripes of reactants separately onto the matrix.Thus each reactant ink is laid down alternately as parallel thin bandsor concentric circles.

Normally, it is desirable to maintain the indicator composition, e.g.dye, for indicating a color change point as one of the separate discreteareas on the test device. For sample testing based on pH changes, it mayalso be desirable to maintain a buffer as a separate area on the testdevice until testing occurs.

It will be obvious to a person skilled in the art that many printingtechniques can find applicability to the present invention. For example,it would be feasible to employ rotogravure printing techniques, silkscreening, and offset printing. Of the foregoing methods, silk screeninghas to date been found to be preferred.

While silk is a preferred material for screening, screens made of othermaterials can also be employed, such as screens of woven polyester,polyamide or metal threads. Usually, art-known silk screens coated withstandard photosensitive resist materials are employed. Afterphotographic exposure of the screen (e.g. in the desired dot pattern)the exposed screen is washed, leaving the exposed pattern for printing.This technique, of course, is not novel and is too well known in theprinting art to require further discussion herein. Preforated sheetsmade of a material such as plastic or thin metal can also be employed. Aperforated sheet can be used in the same manner as a screen by placingthe sheet over the inert matrix, applying reactant to it and drawing adoctor blade across it to spread the reactant material and force itthrough the holes thereby printing the matrix. The screens or sheetsmust be loaded precisely with respect to the inert matrix and theformulation of the reactants must be such that the desired result isachieved without the reactants running together during or afterapplication. Regardless of the method employed, the size of the discretereactant areas applied in known proportions can be varied from verysmall areas, e.g. microdot size, to relatively large size. The alignmentof the printing apparatus obviously becomes more critical as thereactant areas become smaller and closer together.

The printing techniques described herein can be employed with any ofconventional inert matrices used heretofore in diagnostic test devices,such as paper, plastic, and combinations thereof. The particular inertmatrix chosen must be one which adequately reflects incident light sincetest devices are read by visually judging the intensity of reflectedlight from such devices. Optically transparent matrix materials such asTrycite® polystyrene film made by Dow Chemical Co. may be employed.

If desired, paper used as a matrix can be coated to improve its lightscattering efficiency and the printability of the paper, i.e. adherenceof reactants. The surface of the paper can be white in color to reflectas much visible light of all wavelengths as possible. Obviously, a matfinish is preferred over a high gloss finish.

A plastic matrix can have essentially the same optical characteristicsas noted for paper. While plastic has the advantage of being lesschemically reactive and more uniformly reflective, adherence ofreactants can be somewhat more difficult on plastic than paper using theprinting techniques described herein. If desired a white pigment can beincorporated with the plastic to achieve a desired reflective surface.

Known diluent substances useful to reduce hygroscopicity of reactants,such as chloroform, carbon tetrachloride, benzene, and the like; as wellas known wetting agents, such as diglycol laurate, organic phosphateesters of anionic detergents in ethanol and the like, which aid inproducing an even diffusion of color on a test device can beincorporated into the printed reactant compositions of this invention.

Test devices in accordance with this invention can advantageously bemade in the form of long strips or tapes that are rolled up and insertedin a suitable roll-tape dispenser as well as being cut into individualtest strips.

The following examples are presented in order to more clearly describethe invention and to point out preferred embodiments. They are not,however, intended in any way to limit the present invention and are notto be thus interpreted.

EXAMPLE 1 ALTERNATE STRIPES

Two reactant inks were prepared as follows:

Polymer solution: A solution of cellulose acetate in acetone wasprepared. This solution served separately as the vehiclw for eachreactant.

Reactant 1:

In 65 ml of water was dissolved 2.8 g sodium citrate and 4.7 g citricacid. Next was added 50 mg of Tetrabromophenol Blue and 30 ml ofmethanol. The pH was then adjusted to 3.3 by the addition of a buffer.10 ml of the resulting mixture was added to 20 ml of the polymersolution to produce a first reactant ink.

Reactant 2:

2.2 g of sodium citrate and 10 mg of orthocresol sulfonephthaline in 6ml ethanol were added to 24 ml of water and the pH was adjusted to 7.8through the addition of a buffer. 10 ml of this mixture was added to 20ml of the polymer solution to form a second reactant ink.

A small dispensing head was prepared for applying the two inks to apolystyrene matrix. The dispensing head was provided with 14 channelsapproximately 0.03 inch in width. Two dispensing ports fed alternatesets of channels. Hence, the first port provided one ink sample to oddchannels 1,3,5...13, the second provided the other ink sample to evenchannels 2,4,6...14. A portion of each ink was added to its respectiveport and the dispensing head was drawn across a white polystyrenematrix, thus depositing alternating stripes of the first and secondinks. The striped polystyrene matrix was then cut into stripes about 1/4inch in width. These were tested with a 100 mg percent albumin solutionin water and in pure water. The strip which was dipped in the albuminsolution yielded a yellow/green color whereas an identical strip dippedin water became pale yellow.

EXAMPLE 2 Offset Dot Printing (Halftone)

This experiment was performed in order to demonstrate the feasibility ofoffset dot printing of reactants. A rubber plate for the printing waspurchased from a local rubber stamping manufacturer. The manufacture ofrubber plates is well known in the rubber stamp art, and the particularone purchased for this experiment was made using a metallic brass diecontaining normal printing periods as recessed dots. The periods were 30mils in diameter and were squarely arranged in a density of 64 periodsper square inch. An unvulcanized rubber matrix was placed over the dieand pressure was applied causing the rubber to flow into the recesses ofthe metal die. Subsequently, heat was applied to the rubber to vulcanizeit, causing the rubber plate to achieve a permanent configuration. Therubber sheet was then stripped off thus forming the rubber plate.

The rubber plate was cut into two squares, 1/2 inch on the side, thusforming the dies used for printing the reactant dots. Each rubber diewas mounted using rubber cement to the face of an aluminum adapter usedfor mounting the die on a small arbor press which was commerciallypurchased.

Two reactant inks were prepared as in Example 1 for use with the dies.One of the dies was mounted on the press, and a piece of Trycite®polystyrene film obtained from Dow Chemical Co. was mounted in the pressbeneath the die. The die was inked with the first reactant ink and animpression of the ink was made on the polystyrene film. The second diewas then inked with the second reactant ink and mounted in the press.The register of the plastic film was changed such that the dots from thesecond die would be applied in a position juxtaposed with the firstdots. The printing of the second dots completed the preparation of thetest device, which was then air dried.

EXAMPLE 3 Silk Screen Dot Printing

This experiment demonstrates the application of the present invention tothe silk screening printing process. A standard silk screen waspurchased from Dec-O-Art, Inc. in Elkhart, Ind. This screen wasphotographically prepared by that corporation and contained patterns of25 mil dots, 250 per square inch, and 40 mil dots, 125 per square inch.Thus, in the dot patterns on the silk screen, the dots constituted holeswhere an ink could flow through the screen, whereas all other areas inthe dot pattern were closed to ink flow.

A sheet of Trycite® polystyrene film obtained from Dow Chemical Co. wasplaced under the screen at a distance of from 1/16 to 3/16 inches. Aportion of the first reactant ink from Example 1 was then drawn acrossthe screen with the use of a square edged polyurethane squeegee. Byexerting pressure on the squeegee, the screen contacted the polystyreneand a series of dots were laid down approximately the same size as theopenings in the screen. The screen was then cleaned and a second seriesof dots was placed on the polystyrene using the second reactant ink ofExample 1 except that the register of the screen was changed so that thesecond dots were juxtaposed with the first. The printed polystyrenesheet was then dried at ambient conditions, to form a usable printedtest device.

The technique was repeated with the larger dots to likewise yield asatisfactory test device.

From the foregoing, it will be seen that this invention is well adaptedto obtain all of the advantages hereinabove set forth, together withother advantages which are obvious and inherent to the system. Theinvention provides a rapid and relatively inexpensive method of applyingreactants to a test device in a manner which prevents interaction ofreactants until the test device is contacted with a sample to be tested.The shelf life of the test device is significantly improved.

Obviously, many other modifications and variations of the invention ashereinbefore set forth may be made without departing from the spirit andscope thereof.

What is claimed is:
 1. In a method for preparing a dip-and-read testdevice for determining the presence of a constituent in a test sample,whereby said method comprises incorporating a carrier matrix with areagent system comprising first and second reactants, said reactantsmutually interacting in the presence of said constituent to produce adetectable response, the improvement which comprisespreparing a firstreactant ink containing said first reactant, and a second reactant inkcontaining said second reactant, printing said first ink onto saidcarrier matrix in a first array of impressions, and printing said secondink onto said carrier matrix in a second array of impressions, theimpressions of said second array being printed so as to be at leastpartially interspersed with, but substantially not contacting, theimpressions of said first array.
 2. The method of claim 1 in which theinks are printed by offset.
 3. The method of claim 1 in which the inksare printed by screening.
 4. The method of claim 1 in which the inks areprinted by gravure.
 5. The method of claim 1 in which the first ink isprinted in a plurality of parallel stripes, and the second ink islikewise printed in stripes mutually parallel to and positioned betweenthe first ink stripes.
 6. In a method for preparing a dip-and-read testdevice for determining the presence of a constituent in a test sample,whereby said method comprises incorporating a carrier matrix with areagent system comprising first and second reactants, said reactantsmutually interacting in the presence of said constituent to produce adetectable response, the improvement which comprisespreparing a firstreactant ink containing said first reactant, and a second reactant inkcontaining said second reactant, silk screening said first ink onto saidcarrier matrix in a first array of impressions, and silk screening saidsecond ink onto said carrier matrix in a second array of impressions,the impressions of said second array being printed so as to be at leastpartially interspersed with, but substantially not contacting, theimpressions of said first array.
 7. The method of claim 6 in which thefirst and second patterns comprise dots.
 8. The method of claim 6 inwhich the first and second patterns together comprise a plurality ofparallel stripes, the stripes of the first reactant ink beingalternately positioned with respect to the stripes of the secondreactant ink.